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PubMed1999

Quantitative evaluation of heparin-coated versus non-heparin-coated bypass circuits during cardiopulmonary bypass.

The extracorporealization of blood activates various elements of the fibrinolytic, coagulation, and complement systems. It is theorized that advancements in biocompatibility ameliorate many of the changes leading to improved patient management. The purpose of this study was to determine if heparin-coated circuit (HCC) utilization during cardiopulmonary bypass enhances patient outcomes in a cost-effective manner. A search of the English medical literature was completed to identify all clinical, prospective, randomized trials comparing HCC and non-HCC in patients undergoing coronary artery bypass grafting or valvular surgery. Twenty-six papers consisting of a sample size of 1515 patients were identified and included in the study parameters. The study distinguished between Duraflo II and Carmeda coating techniques and matched papers with different heparin loading doses, as well as use of a heparin-coated cardiotomy. Study parameters were matched for all papers and analyzed according to the availability of data. Statistically significant benefits of HCC were found in postoperative blood loss, time in the ICU, end bypass C3a, time to extubation, end bypass lactoferrin, and end platelet count, but not with respect to postoperative chest tube drainage, red blood cell transfusions, and end bypass TAT complex, D-dimers, and BTG. Data comparing the use of coated or uncoated cardiotomy utilization failed to demonstrate a benefit to heparin coating. Several immunological variables were ameliorated when Carmeda HCC was utilized, although data were insufficient to establish a cost-benefit analysis. In conclusion, heparin-coated circuitry provided statistically better results when compared to noncoated circuitry.

Cardiopulmonary Bypass↗
PubMed1999

Alterations in C3 activation and binding caused by phosphorylation by a casein kinase released from activated human platelets.

A casein kinase released from activated human platelets phosphorylates a number of plasma proteins extracellularly, and that activation of platelets in systemic lupus erythematosus patients parallels an increase in the phosphate content of plasma proteins, including C3. The present study was undertaken to characterize this platelet protein kinase and to further elucidate the effect(s) on C3 function of phosphorylation by platelet casein kinase. The phosphate content of human plasma C3 was increased from 0.15 to 0.60 mol phosphate/mol of C3 after platelet activation in whole blood or platelet-rich plasma. The platelet casein kinase was distinct from other casein kinases in terms of its dependence on cations, inhibition by specific protein kinase inhibitors, and immunological reactivity. C3 that had been phosphorylated with platelet casein kinase was tested for its susceptibility to cleavage by trypsin or the classical and alternative pathway convertases and its binding to EAC and IgG. Phosphorylation did not affect the cleavage of C3 into C3a and C3b, but the binding of fragments from phosphorylated C3 to EAC14oxy2 cells and to IgG in purified systems and in serum was increased by 1.6-4.5 times over that of unphosphorylated C3. A covariation was seen between the enhanced binding of C3 fragments to IgG after phosphorylation and an increased ratio of glycerol/glycine binding, from 2.0 for unphosphorylated C3 to 4.9 for phosphorylated C3. The present study suggests that an overall effect of phosphorylation of C3 by platelet casein kinase is to enhance the opsonization of immune complexes.

Animals↗
PubMed1988

Differential neutrophil activation before and after endotoxin infusion in enterally versus parenterally fed volunteers.

This study was done to determine whether or not increased susceptibility to infection seen in enterally versus parenterally fed patients was caused by altered neutrophil (PMN) responsiveness. To determine the differential effects of route of feeding on human PMN activation, plasma C3a levels, circulating PMN counts, PMN migration to leukotriene B4 (LTB4), the peptide, N-formyl-methionyl-leucyl-phenylalanine (FMLP) and zymosan activated serum (ZAS) and generation of LTB4 were assayed before and after and infusion of endotoxin. Nine normal volunteers were enterally (n=4) or parenterally (n=5) fed a diet sufficient to maintain body weight for seven days prior to a standard challenge of endotoxin. Samples were taken prior to the infusion and hourly thereafter for six hours. Prior to the injection of endotoxin, significant differences were seen in the two feeding groups. Plasma C3a levels, absolute circulating PMN counts and chemotaxis to LTB4 were all significantly (p less than 0.02) elevated in the enterally fed group. Generation of LTB4 was higher in the intravenously fed group at base line than the orally fed group (p less than 0.05). Plasma C3a levels rose in the enterally fed group, but not in the intravenously fed group, at two hours after infusion. Neutrophil counts rose in both feeding groups after endotoxin infusion; but the change in percentage was greater in the enterally fed group than in the intravenously fed group. Chemotaxis to FMLP and ZAS was not different during the study and did not differ between the two feeding can have significant impact on neutrophil function and that parenteral nutrition may impair host responsiveness.

Chemotaxis↗
PubMed1997

Cytokine profiles during clinical high-flux dialysis: no evidence for cytokine generation by circulating monocytes.

Secretion of cytokines by monocytes has been implicated in the pathogenesis of dialysis-related morbidity. Cytokine generation is presumed to take place in two steps: induction of mRNA transcription for cytokines by C5a and direct membrane contact, followed by lipopolysaccharide (LPS)-induced translation of mRNA (priming/second signal theory, Kidney Int 37: 85-93, 1990). However, the in vitro conditions on which this theory was based differed markedly from clinical dialysis. To test this postulate for routine hemodialysis, 13 patients were studied cross-over with high-flux cuprammonium (CU), cellulose triacetate (CTA), and polysulfon dialyzers, using standard bicarbonate dialysate, as well as CTA with filtered dialysate (fCTA). Besides leukocytes, C3a, C5a, and limulus amebocyte lysate reactivity, tumor necrosis factor (TNF)-alpha, interleukin (IL)-1 beta, IL-6, IL-1RA, soluble TNF receptors, and IL-1 beta mRNA were assessed. Only during dialysis with CU did C5a increase significantly (561 to 8185 ng/ml, P < 0.001). Endotoxin content of standard bicarbonate was higher than filtered dialysate (median, 24.3 and < 5 pg/ml respectively, P = 0.002), whereas limulus amebocyte lysate reactivity was not detected in the blood, except in the case of CU. TNF-alpha levels were elevated before, and remained stable during, dialysis, independent of the modality used. IL-1 beta, IL-6, and mRNA coding for IL-1 beta could not be demonstrated. IL-1RA and soluble TNF receptors (p55/p75) were markedly elevated compared with normal control subjects, but showed no differences between fCTA and CTA. To summarize, no evidence was found for production and release of cytokines by monocytes during clinical high-flux bicarbonate hemodialysis, neither with complement-activating membranes nor with unfiltered dialysate. Therefore, this study sheds some doubt on the relevance of the "priming/second signal" theory for clinical practice. The data presented suggest that reluctance to prescribe the use of high-flux dialyzers, as advocated in many reports, may not be warranted.

Adult↗
PubMed1988

Structural characterization of the C4a anaphylatoxin from rat.

The C4a anaphylatoxin was purified from rat sera activated by heat-aggregated IgG. The anaphylatoxin was isolated by a three-step purification procedure and was judged to be homogeneous based on visualization of a single stained band after electrophoresis on both cellulose acetate membrane strips and on 9% SDS-polyacrylamide gels. Results from Ouchterlony and radioimmunoassay analysis indicated that neither rat C5A nor C3a contaminated the C4a preparation. Rat C4a is a glycoprotein estimated to be 11,000-12,000 mol. wt and contains 76 amino acid residues representing a mol. wt of 8577 and one oligosaccharide unit of 2000-3000 mol. wt. Rat C4a is weakly active in contracting guinea pig ileum at 0.1-1 microM, which is comparable with the activity of human C4a. Both human and bovine C4a are polypeptides free of carbohydrate while rat and presumably mouse C4a are glycoproteins. The complete primary structure of rat C4a anaphylatoxin has been elucidated as follows: (formula; see text)

Amino Acid Sequence↗
PubMed1998

Human anaphylatoxin C4a is a potent agonist of the guinea pig but not the human C3a receptor.

The interaction of human anaphylatoxin C4a with the guinea pig (gp) and human (hu) C3a receptors (C3aR) was analyzed using human rC4a, which exhibited C4a-specific activity on guinea pig platelets. A gpC3aR of 475 residues with a large second extracellular loop and a peptide sequence approximately 60% identical to the huC3aR was isolated from a genomic DNA library and found to be expressed in guinea pig heart, lung, and spleen. HEK-293 cells cotransfected with this clone, and a cDNA encoding G alpha-16 specifically bound (Kd = 1.6+/-0.7 nM) and responded functionally to C3a with an intracellular calcium mobilization (ED50 = 0.18+/-0.02 nM). Human rC4a weakly bound to both the hu- and gpC3aR (IC50 > 1 microM). However, only HEK-293 cells expressing the gpC3aR responded functionally to rC4a (ED50 = 8.7+/-0.52 nM), while cells expressing the huC3aR did not (c < or = 1 microM). Thus, through an interaction with the C3aR, huC4a may elicit anaphylatoxic effects in guinea pigs but not in man.

Amino Acid Sequence↗
PubMedDate not supplied

Clinical effects of a polyethylene glycol grafted cellulose membrane on thrombogenicity and biocompatibility during hemodialysis.

The biocompatibility and thrombogenicity of polyethylene-glycol (PEG)-grafted cellulose hemodialysis (HD) membranes (PEGC) were investigated in cross-over HD of five HD patients with ordinary cellulose (OC). The PEGC significantly suppressed transient leukocyte and thrombocytopenia, and release of C3a, beta-thromboglobulin and platelet factor 4, in corresponding with the quantity of grafted PEG. HD with PEGC resulted in lower granulocyte elastase production, protein and blood cells adsorption on the membrane surface than those with OC. Minimum heparin in HD with PEGC was three times lower than that with OC, with the thrombin-antithrombin III complex elevation lower than that in HD with OC. The results indicate that the grafted PEG effectively suppresses blood and membrane interaction, thus improving biocompatibility and reducing thrombogenicity in clinical HD.

Biocompatible Materials↗
PubMed1989

Monitoring of the inflammatory response in early peritonitis.

The inflammatory response which is similar in all forms of peritonitis was recorded by determining the levels of parameters shown to represent the activation state of plasmatic and cellular systems as well as the inhibitory capacity of the plasma. In a selected series of patients with different underlying diseases, blood sampling was started at the time of admission when the clinical finding of an acute abdomen led to emergency laparotomy. Depending upon the duration of the illness and the severity of the peritonitis, a significant increase in fibrinopeptide A and of C3a could be detected within a few hours, which was followed by an increase in the elastase alpha-1-proteinase inhibitor complex. Differences due to variable cause could not be found. There was a striking correlation between the preoperative values of these three parameters and the postoperative course of the patients. Additionally, there was a significant enhancement of an endothelial proliferation-inhibiting capacity in the serums of the lethal group, whereas endotoxin could only be detected in trace amounts in four patients with intraabdominal infection in the preoperative period.

Adult↗
PubMed1984

A prospective clinical and immunologic analysis of patients with serum sickness.

We prospectively evaluated the clinical and immunologic features of serum sickness in 12 patients with bone-marrow failure treated for 10 days with intravenous infusions of horse antithymocyte globulin. Eleven of the 12 patients had signs and symptoms of serum sickness 8 to 13 days after beginning therapy with antithymocyte globulin. Eleven patients (including 10 of 11 with signs and symptoms of serum sickness) acquired circulating immune complexes, with peak levels occurring at 10 to 12 days. Serum C4 and C3 levels fell precipitously, with nadirs on the 10th day. Plasma levels of C3a anaphylatoxin were elevated in the four patients in whom it was measured. Eight of the 11 patients with signs and symptoms of serum sickness had a characteristic serpiginous erythematous and purpuric eruption on the hands and feet at the junction of palmar and plantar skin. Direct immunofluorescence of skin lesions revealed immune deposits (IgM, IgE, IgA, or C3) in the blood vessels of three of five patients. This study documents the immunopathology of serum sickness in human beings and describes a cutaneous marker for the disease.

Adolescent↗
PubMed1980

Suppression by prostaglandin E1 of vascular permeability induced by vasoactive inflammatory mediators.

Systemic treatment of rats with prostaglandin E1 or with its stable derivative, 15-(S)-15-methyl-Prostaglandin E1, markedly reduce the increases in vasopermeability induced by the intradermal injection of histamine, serotonin, bradykinin, C3a, and compound 48/80. This inhibition is dependent on the dose of the vasopermeability factor and the duration of time between injection of the prostaglandin and intradermal injection of vasopermeability factor. The inhibition of associated ultrastructurally with a preservation of tight junctions between endothelial cells. The structure-function specificity of the inhibitory effects of the prostaglandin is shown by the diminished effects of prostaglandin A2, and the lack of inhibition by prostaglandin F2 alpha. These data provide evidence that PGE1 interferes with the local effects of vasopermeability mediators and may explain the earlier observations at PGE1 protects animals from immune complex-induced nephritis.

Animals↗
PubMed1971

The antigenic and molecular alterations of C3 in the fluid phase during an immune reaction in normal human serum. Demonstration of a new conversion product, C3x.

During the reaction of an immune precipitate with fresh human serum, C3 undergoes a number of molecular alterations with the formation of conversion products differing from those obtained when purified components react. Those products which remain in the fluid phase, the subject of the present paper, have been identified by their reaction with monospecific antisera to the three antigenic determinants of C3, A, B, and D, after electrophoresis in agar or polyacrylamide gel. When purified C3 reacts with EAC1,4,2, C3i is found in the fluid phase. C3i, a loose complex of C3a and C3b, is in a conformational state whereby only the A and D antigens, present on its C3b portion, will consume antibody. The B antigen, present on the C3a portion of C3i, is unavailable for combination with antibody until C3i dissociates. In the fluid phase of the reaction of an immune precipitate with whole serum, C3i, C3a, and C3b, formed when purified components react, cannot be found. Instead the end products of the reaction appear to be C3c, which contains the A antigen, and C3d, which contains the D antigen. C3c and C3d are similar to the beta1A and alpha2D produced by the aging of serum but differ in their mobilities in acrylamide gel and in agar. The C3c and C3d generated by an immune precipitate also differ slightly from the C3c and C3d produced by the reaction of trypsin with C3 in whole human serum. As human serum reacts with an immune complex, native C3 appears to undergo a primary alteration before conversion. This alteration results in a molecular species of C3 which is labile at 56 degrees C for 30 min, fails to expose additional A and D antigenic sites upon aging, and which forms beta1A and C3d rather than beta1A and alpha2D during aging. In addition to this altered form of native C3, a new conversion product, C3x, is formed as whole serum reacts with an immune complex. C3x is not found in systems utilizing pure complement components. C3x is like C3 in that it bears all three antigenic determinants but differs in that it has a slightly faster mobility in polyacrylamide gel than does native C3. C3x is not only found in the fluid phase but is also bound to the immune precipitate. Finally, the fluid-phase kinetics of each of the antigens of C3 have been determined as normal human serum reacts with an immune precipitate. These illustrate that nearly the entire population of native C3 molecules undergoes conversion rapidly as manifested by the disappearance of the B antigen from the fluid phase. Moreover, the kinetics of the fluid-phase A and D antigens reflect that the conversion of C3 in serum is quantitatively not the same as when purified C3 reacts with C4,2.

Adult↗
PubMed1999

Cloning and characterization of a rabbit ortholog of human Galpha16 and mouse G(alpha)15.

A cDNA was cloned from a rabbit spleen cDNA library which encoded a G-protein alpha subunit peptide of 374 amino acids, that at the peptide level exhibited 86% and 79% identity with human Galpha16 and mouse G(alpha)15, respectively. The rabbit G(alpha)subunit cDNA was subcloned into a mammalian expression vector and transiently co-transfected into HEK-293 cells along with cDNAs encoding the human C3a, C5a, or nociceptin/orphanin FQ receptors. In all three cases the rabbit G alpha subunit behaved similarly to G(alpha)15 or G(alpha)16 and effectively coupled the transfected receptors to intracellular calcium mobilization pathways. By nucleotide sequence homology and functional activity the rabbit G(alpha) subunit appears to be the ortholog of human G(alpha)16 and mouse G(alpha)15.

Amino Acid Sequence↗
PubMed2003

Nuclear factor kappaB mediates a procoagulant response in monocytes during extracorporeal circulation.

OBJECTIVE: The objective of this study was to examine the mechanism of procoagulant activity and inhibition in whole blood during extracorporeal circulation. METHODS: In this study we examine the development of procoagulant activity and monocyte activation in heparinized whole blood passing through a closed circuit consisting of a pump and silicone envelope membrane oxygenator for 6 hours. RESULTS: Anaphylatoxins, C3a and C5a, determined by means of enzyme-linked immunosorbant assay, appeared in the blood within 30 minutes of circulation. Circulated blood developed a marked potential for coagulation demonstrated in a 1-step clotting assay that reached maximal activity by 4 hours of circulation. This procoagulant activity was neutralized by anti-tissue factor antibody, suggesting a prominent role for the extrinsic pathway in pump-induced intravascular coagulation. Isolation of monocytes from circulated blood revealed that tissue factor expression is upregulated on the cell surface. Furthermore, we observed nuclear factor kappaB nuclear translocation in monocytes from blood passing through the circuit, suggesting that tissue factor expression was due to monocyte stimulation and transcriptional activation of the tissue factor gene. Tissue factor expression resulted in an approximately 30-fold increase in thrombin generation. Monocyte nuclear factor kappaB activation, monocyte tissue factor expression, thrombin generation, and the procoagulant activity of blood in extracorporeal circulation were all blocked by the proteasome inhibitor MG132. CONCLUSIONS: We conclude that intravascular tissue factor expression during extracorporeal circulation of blood is due to nuclear factor kappaB-mediated activation of monocytes (possibly by complement), which can be controlled pharmacologically.

Blood Coagulation↗
PubMed1989

Leukotriene formation by human polymorphonuclear leukocytes from endogenous arachidonate. Physiological triggers and modulation by prostanoids.

Human polymorphonuclear leukocytes (PMN) were isolated from freshly drawn venous blood by Dextran sedimentation and discontinuous Percoll gradient centrifugation. The effects of several putative triggers of the leukotriene formation such as C5a, PAF, FMLP, C3a, PMA, LTC4, LTD4, LTB4 or arachidonate were studied by RP-HPLC analysis. 280 nM C5a, 100 nM FMLP, 1 microM PAF or 20 microM arachidonate induced a marginal formation of 1.5-18 ng of LTB4 plus LTB4 metabolites/2 x 10(7) PMN. 560 nM C3a, 100 nM PMA, 1 microM LTC4, 1 microM LTD4 and 1 microM LTB4 each failed to induce any formation of 5-lipoxygenase products. Pretreatment of the cells with 40 microM ethylmercurithiosalicylate (merthiolate) enhanced the leukotriene formation by 100 nM FMLP about 40-fold, by 280 nM C3a about 120-fold and by 1 microM PAF about 14-fold. Merthiolate itself induced no leukotriene formation from human PMN and reduced the leukotriene formation by 20 microM arachidonate. The FMLP/merthiolate-induced activation of the PMN was concentration-dependent in respect to both FMLP and merthiolate. 1 microM LTC4, 1 microM LTD4 or 1 microM LTB4 also failed to trigger any LTB4 formation of merthiolate-treated PMN. 560 nM C3a or 100 nM PMA in combination with 40 microM merthiolate induced a slight formation of 28 ng and 10 ng of LTB4 plus LTB4 metabolites, respectively. The FMLP/merthiolate-induced leukotriene formation was modulated by prostanoids. PGE2, PGE1, PGD2 and 6-keto-PGE1 each evoked a concentration-dependent inhibition of the leukotriene formation with IC50 values of 0.07 microM, 0.18 microM, 0.27 microM and 6 microM respectively. In addition, significant inhibitory effects by PGI2, Iloprost (a carbacyclin analogue of prostacyclin), PGF2a or 6-keto-PGF1a were achieved; the corresponding IC50 values, however, amounted to 19-59 microM. Thus these compounds were about 500-fold less potent in comparison with PGE2 in inhibiting LTB4 formation by human PMN.

Arachidonic Acids↗
PubMed1989

[Effectiveness and tolerance of the C3 convertase inhibitor, N-acetyl-aspartyl-magnesium glutamate with anti-allergic action. Results of a double-blind study].

N-Acetyl-aspartyl magnesium glutamate (Rhinaaxia, NAAGA) is a topically active antiallergic dipeptide. The compound acts in two different ways. On the one hand NAAGA inhibits the mast cell-degranulation, on the other hand this compound blocks the activation of the C3-convertase, subsequently followed by a blocked cleavage of the fragments C3a and C5a, respectively. 20 patients suffering from pollinosis were treated for 2 weeks according to a randomized double-blind placebo-controlled study. Besides subjective complaints nasal obstruction was objectively documented via rhinomanometria. 9 out of 10 patients under placebo had to use the rescue drug tritoqualine, a histidine decarboxylase inhibitor, compared to none in the verum group (p less than 0.01). After 14 days of treatment with NAAGA the nasal peak flow rate increased by 21.5 l/min and 21.8 l/min in the tritoqualine/placebo group, respectively (not significant). Nasal obstruction improved statistically significantly after 7 and 14 days of treatment in both groups. Tolerance was reported to be good in either group.

Adolescent↗
PubMed1991

Regulation of mediator release by human basophils: importance of the sequence and time of addition in the combined action of different agonists.

Biologically active molecules affecting basophil function can be divided into 4 groups according to their capacity to induce basophil degranulation and/or leukotriene generation: (1) full agonists such as anti-IgE or fMLP, which induce both histamine and leukotriene release; (2) partial agonists such as C5a, which induces degranulation only; (3) incomplete agonists such as neutrophil-activating peptide-1, platelet-activating factor or C3a, which induce mediator release only after cytokine preincubation, and (4) basophil response modifiers, such as interleukin-3, interleukin-5 and granulocyte/macrophage- colony-stimulating factor, which (a) enhance the releasability to all basophil agonists, (b) change the mediator profile, (c) enhance the rate of mediator release, (d) render basophils responsive to lower agonist concentrations and (e) render basophils responsive to incomplete agonists. We demonstrated that histamine release and de novo synthesis of lipid mediators are clearly separately regulated, and that combined actions of different molecules are of importance. In particular, the type(s), the time interval and the sequence of action of basophil-activating molecules are crucial for the final outcome of the basophil release reaction.

Basophils↗
PubMed1984

Natural release of covalently bound C3b from cell surfaces and the study of this phenomenon in the fluid-phase system.

Covalently bound C3b is released from cell surfaces (EAC1423 and zymosan-C3b) on incubation under physiologic conditions. The release of C3b from cell surfaces occurs by the cleavage of the covalent bond. Sodium dodecyl sulfate (SDS) abolishes the release, thereby indicating the requirement of the native structure of C3b in this process. The phenomenon of release of C3b from cell surfaces has also been observed in the fluid-phase system by using C3b-[3H]glycerol. The kinetics of the release of [3H]glycerol from C3b-[3H]glycerol were studied at 37 degrees C in 0.15 M phosphate buffer, pH 7.4. The first-order rate constant was found to be 0.028 +/- 0.003 hr-1. The release does not take place in either 8 M urea or 6 M guanidine hydrochloride, at pH 7.4. Under alkaline conditions, the rate of release is unaffected in the presence of SDS, indicating that the release in this pH range is not dependent on the native structure of the protein. From the Arrhenius plot in the temperature range 18 to 37 degrees C, an apparent activation energy for the hydrolysis reaction of 21.2 kcal/mol was calculated. The release phenomenon is exclusive for ester-linked complexes, as inferred by the absence of release of [3H]threonine from C3b-[3H]threonine, wherein the linkage is of the amide type. The presence or absence of the C3a portion of the molecule has no effect on the rate of release. The modification of the -SH group of C3i-/C3b-[3H]glycerol alters the rate of hydrolysis of the ester bond between C3i/C3b and [3H]glycerol. Protease inhibitors (PMSF, benzamidine HCl, and DFP) do not alter the rate of release, indicating that the hydrolysis reaction is not due to trace amounts of contaminating proteases. Thus, it appears that some chemical group(s) of C3i/C3b is (are) involved in the intramolecular hydrolysis of the ester bond between C3i/C3b and small molecules. This phenomenon may play an important role in the release of C3b from receptive surfaces once the biologic functions that require covalently bound C3b have been mediated.

Alkylation↗
PubMed1991

Proteolysis of C3 on U937 cell plasma membranes. Purification of cathepsin G.

Covalent binding of C3 fragments to U937 cell membranes involved a cell surface-associated proteolytic activity. Two proteases able to cleave C3 were purified from U937 plasma membranes. Purification involved solubilization of the membranes and ion exchange chromatography. One of the purified proteases was identified as elastase, based upon a substrate specificity for benzyloxycarbonylalanine-o-nitrophenyl ester and complete inhibition by elastatinal and methoxysuccinyl-alanyl-alanyl-prolyl-valyl-chloromethyl-ketone. The other protease (m.w. 28,000) is cathepsin G, as deduced from the amino acid composition, the amino-terminal sequence, and the substrate specificity for succinyl-alanyl-alanyl-phenylalanine-p-nitroanilide. These two lysosomal proteases are present on the U937 cell surface, as confirmed by immunofluorescence analysis. Plasma membrane elastase and cathepsin G from U937 cells cleave C3 into C3a- and C3b-like fragments; further incubation leads to C3c- and C3dg-like fragments, as judged from SDS-PAGE analysis of the digests. Sequencing of the C3b-like fragment purified by reverse phase chromatography indicates that initial cleavage of C3 by purified cathepsin G occurs at two positions in the amino-terminal part of the alpha-chain, at a Arg-Ser bond located between residues 748 and 749 and at a Leu-Asp bond between residues 751 and 752. These proteases are, thus, able to generate, on the U937 surface, active fragments of C3, which are likely to be involved in cell-protein and cell-cell interactions.

Amino Acid Sequence↗

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